Treatment of [3H]-choline- or [14C]-ethanolamine-labelled undifferentiated bipolar and differentiated multipolar CG-4 line oligodendrocytes with 12–0-tetradecanoylphorbol 13-acetate (TPA) to activate protein kinase C stimulated the release of choline or ethanolamine metabolites to the medium over controls. Ro31–8220, a PKC inhibitor, reduced TPA-stimulated release of choline- and ethanolamine-metabolites to basal levels. TPA treatment of both bipolar and multipolar cells caused rapid contraction of processes leaving rounded up cells: this effect was blocked by Ro31–8220. After 12–15 h exposure to TPA, bipolar undifferentiated CG-4 line cells extended short processes again and the cells became multipolar. Nocodozole, an agent which disrupts microtubules and caused CG-4 line cells to round up, caused increased choline or ethanolamine-metabolite release to the medium over basal levels suggesting that some release during TPA-treatment might occur due to process fragmentation. However, the transphosphatidylation reaction confirmed that phospholipase D was active in these cells. Exposure of bipolar undifferentiated CG-4 line cells to TPA resulted in down-regulatation of PKC-α and PKC-βΙΙ which could not be detected by Western blotting after a few hours; PKC-ε was down-regulated much more slowly but PKCs δ, ζ and ι were not influenced by 48 h exposure of cells to TPA. Formation of phosphatidylethanol in the transphosphatidylation reaction was markedly reduced in TPA down-regulated cells indicating a role for PKCs α and βΙΙ in phospholipase D activation in CG-4 line oligodendrocytes.